Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system
Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system
复制标题
利用 CRISPR/Cas9 系统对棉铃虫中钙粘蛋白作为 Bt 毒素 Cry1Ac 受体的功能进行验证
DOI：
10.1016/j.ibmb.2016.06.008
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发表时间：
2016-09-01
影响因子：
3.8
通讯作者：
Wu, Yidong
中科院分区：
文献类型：
--
作者：
Wang, Jing;Zhang, Haonan;Wu, Yidong
钙粘蛋白是苏云金芽孢杆菌（Bt）Cry 1A毒素在包括棉铃虫在内的几种鳞翅目昆虫体内的受体。钙粘蛋白基因HaCad的破坏与H. armigera。利用CRISPR/Cas9基因组编辑系统（CRISPR-associated protein 9，CRISPR/Cas9），从Cry 1Ac敏感的H. armigera被成功击倒。鉴定了具有4个核苷酸的移码缺失的单个阳性CRISPR事件，并使其纯合以产生命名为SCD-Cad的敲除系。Western印迹证实HaCad不再在SCD-Cad系中表达，而210 kDa的完整HaCad存在于亲本SCD菌株中。生物测定结果表明，SCD-Cad对Cry 1Ac的抗性是SCD的549倍，但对Cry 2Ab的敏感性无明显变化。我们的研究结果不仅为HaCad作为Cry 1Ac的功能性受体提供了强有力的反向遗传学证据，而且还表明CRISPR/Cas9技术可以作为一种强大而有效的基因组编辑工具来研究全球农业害虫H. armigera。(C)2016爱思唯尔有限公司版权所有。
Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera. (C) 2016 Elsevier Ltd. All rights reserved.